The development of antibiotics revolutionized the practice of medicine in the second half of the 20th century. Mortality due to infectious diseases decreased markedly during this period. Armstrong et al., (1999) PAMA. 281, 61-66. Since 1982, however, deaths stemming from infectious diseases have steadily climbed in parallel with the rise of antibiotic resistant pathogens. A wide variety of medically important bacteria are becoming increasingly resistant to antibiotics commonly used in the treatment of clinical infections. Thousands of reports and books have appeared in the literature during the past 20 years that document this phenomenon. Armstrong et al., (1999) PAMA. 281, 61-66; Dessen et al., (2001) Curr. Drug Targets Infect. Disord. 1, 11-16; Rapp (2000) Surg Infect (Larchmt). 1, 39-47; Benin & Dowell (2001) Antibiotic resistance and implications for the appropriate use of antimicrobial agents, Humana Press, Totowa, N.J.
While there is a need to teach more appropriate use of antibiotics, more importantly there is a need for new antibiotics. Vancomycin is considered to be the last line of defense against many serious bacterial infections. The finding of vancomycin resistance strains of pathogenic bacteria is alarming; it portends the rise of multidrug resistant pathogens that would be untreatable with currently available drugs. The fear is that we will, in effect, return to the pre-antibiotic era unless new antibiotics are developed soon.
There is a small, structurally novel class of antibiotics called lantibiotics (Class I bacteriocins) which can be divided into 5 subclasses based on differences in their chemistry and biosynthesis: Type A(I), Type A(II), Type B, Two-Component and those of unknown structures. This class of antibiotics has been known for decades but has not been extensively tested for their potential usefulness in treating infectious diseases even though many lantibiotics are known to be both potent and have a broad spectrum of activity, notably against gram positive species. The principal reason for this is the general difficulty of obtaining these molecules in sufficient, cost effective amounts to enable their testing and commercialization.
Nisin A (FIG. 1) provides a good example of a lantibiotic, and of the number and types of chemical complexities associated with lantibiotics. Lantibiotics are rich in the sulfur-containing amino acids, lanthionine (Lan, ala-5-ala) and, frequently, 3-methyl-lanthionine (MeLan, abu-5-ala). Lan consists of alanine residues that are connected via thioether bridges to create ring structures that are critical for bioactivity. Typically there are 3-5 such rings on a lantibiotic, and often many of the rings overlap with each other. Lan and MeLan are believed to invariably have the meso-stereochemistry. In addition to the Lan and MeLan residues, there may be other post-translationally modified amino acids (FIG. 2) found in lantibiotics, such as 2,3-didehydroalanine (Dha), 2,3 didehydrobutyrine (Dhb), unsaturated lanthionine derivatives such as S-amino vinyl-D-cysteine (AviCys) and S-amino-D-methylcysteine, as well as D-alanine, 2-oxopropionyl, 2-oxobutyryl, and hydroxypropionyl residues. As in the case of Nisin A, the ring structures made by Lan and MeLan may be overlapped (e.g., rings D and E), further adding to the complexity of the molecule.
Gram positive bacteria are responsible for biosynthesis of the known lantibiotics. They make the mature molecule using a series of sequential enzymatic steps that act on a ribosomally synthesized prepropeptide. The genes responsible for encoding the modifying enzymes are typically clustered on an 8-10 Kb DNA fragment that may reside on the chromosome, a plasmid, or as part of a transposon. In Type A(I) lantibiotics, all the serine and threonine residues in the ribosomally synthesized prepeptide encoded by the lanA gene are dehydrated by an enzyme encoded by the lanB gene and these dehydrated amino acids are involved in the formation of thioether linkages to a nearby cysteine residue that is situated more toward the carboxyl end of the molecule. This reaction is catalyzed by the protein expressed by the lanC gene. In the case of certain lantibiotics, such as epidermin and mutacin 1140, the C-terminal cysteine is decarboxylated by the enzyme expressed by the lanD gene and converted into an S-amino vinyl-D-cysteine. Following transport out of the cell by the product of the lanT gene, the leader sequence of the modified prepropeptide is then cleaved by an extracellular protease encoded by lanP to produce mature antibiotic. Ra et al., (1996) Microbiology-Uk. 142, 1281-1288; Kupke & Gotz (1996) Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology. 69, 139-150; Kuipers et al., (1996) Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology. 69, 161-169.
Attempts to study lantibiotics for their potential usefulness in therapeutic applications have been hindered by the difficulty of obtaining them in sufficient amounts or with sufficient purity. Of the 40 or so lantibiotics characterized to date (Chatterjee et al., (2005) Chemical Reviews. 105, 633 683) only the Type A(I) lantibiotic, Nisin A, produced by Streptococcus lactis, has been made in commercial quantities, and it has found wide application as a food preservative for the past 50 years. The long-term, widespread use of Nisin A without the development of significant resistance (DelvesBroughton et al., (1996) Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology. 69, 193-202) has provided a strong impetus to develop additional lantibiotics for various applications.
Large scale production of Nisin A is performed using a fermentation process that has been refined over the years. A purification protocol for Nisin A has recently been filed as a US patent (USPA 2004/0072333). The protocol utilized a cocktail of expensive proteases followed by column chromatography. However, there is no published, commercially viable procedure for the purification of Nisin A. This demonstrates the current interest in finding an adequate method of producing pure Nisin A and other lantibiotics for therapeutic applications.
Various potential options present themselves for large scale production of lantibiotics. From the standpoint of cost of materials, fermentation processes unarguably would be the best method. Current fermentation methods for many lantibiotics yield microgram per liter quantities, which is not sufficient for drug development.
Alternatively, in vitro production utilizing the lantibiotic modification machinery has been explored in Type A(I) lantibiotics. Kupke & Gotz (1996) Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology. 69, 39-150; Kuipers et al., (1996) Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology. 69, 161-169. The enzymes responsible for post-translational modification of the lantibiotic prepropeptide are not active in cell-free lysates or as purified entities, with the exception of LanD. Kupke & Gotz (1996) Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology. 69, 139-150; 10; Kupke & Gotz (1997) Journal of Biological Chemistry. 272, 4759-4762; Kupke et al., (1992) Journal of Bacteriology. 174, 5354-5361; Kupke et al., (1993) Fems Microbiology Letters. 112, 43-48; Kupke et al., (1995) Journal of Biological Chemistry. 270, 11282-11289; Kupke et al., (1994) Journal of Biological Chemistry. 269, 5653-5659. In the case of Type A(II) lantibiotics, it has been recently reported in Science, that in vitro synthesis of lacticin 481 is possible. Molecules belonging to this group and Type B lantibiotics use only a single multiheaded enzyme, LanM, to accomplish the formation of the Dha, Dhb, Lan, and MeLan residues. Xie et al., (2004) Science. 303, 679-681. The report of lacticin 481 biosynthesis did not provide any detailed information regarding yield or purity, but their work was performed on the nanogram scale. The progress described in this report represents a small but significant step forward, and its widely acclaimed reception further points to the pressing need for the development of lantibiotics as therapeutic agents.
A third option for commercial scale production of lantibiotics using the lan gene cluster cloned into appropriate expression vector(s) and a non-sensitive host is unlikely due to the complexity of the system and the likely need for differentially regulating expression of the various genes involved. The lan gene cluster for gallidermin has been cloned into Bacillus subtilis in an attempt to improve production of this particular lantibiotic. However, this strategy did not result in greatly increased yields and will not be suitable for all lantibiotics since gene regulatory sites are known to vary from species to species. A related approach made use of an artificial gene for mutacin 1140 cloned into Escherichia coli. This artificial gene replaced the natural codons for the serine and threonine residues involved in thioether bridge formation with cysteine codons. This modified gene was cloned in pET32 and expressed in the Origami strain of E. coli to maximize disulfide linkages. Novel chemical methods were developed to extrude a single sulfur atom from the disulfide groups thereby converting them to thioethers. In general, this method proved feasible, but the yields obtained were low owing to the multiple permutations of disulfide bonds and the difficulty in separating out the active form from non-active isomers.
Critical to the bioactivity of Nisin A and other lantibiotics are the often overlapping ring structures, creating a difficult problem to overcome synthetically. In vitro synthetic methods have been widely investigated for the synthesis of various lanthionine containing bioactive peptides as well as lantibiotics. The challenge of synthesizing lantibiotics is arduous and, thus far, no comprehensive synthetic strategy has evolved. Several methods of synthesizing lanthionines have been reported in the literature. These include the in situ-based desulfurizations of cystine units in preassembled peptides using basic or nucleophilic conditions. Galande et al., (2003) Biopolymers (Peptide Science) 71, 543-551; Galande & Spatola (2001) Letters in Peptide Science. 8, 247-251. The methods of desulfurizations are yet to show any commercial viability due to lack of diastereoselectivity and poor yields. Biomimetic approaches have also been used where Dha residues are generated in a preformed peptide followed by a Michael addition to form the lanthionine ring. The preorganization of the peptide presumably leads to a diastereoselective Michael addition. Burage et al., (2000) Chemistry A European Journal. 6, 1455-1466. Peptide cyclization on oxime resin has also been employed wherein a linear peptide containing an orthogonally protected lanthionine is synthesized followed by cyclization and cleavage of the cyclic peptide product. Melacini et al., (1997), J. Med. Chem. 40, 2252-2258; Osapay et al., (1997) Journal of Medicinal Chemistry. 40, 2441-2251. These methods are promising but lack the ability to produce lantibiotics with overlapping thioether rings. This becomes particularly important when one takes into account that most of the known lantibiotics contain overlapping rings.
Conceptually, there are clear advantages to developing in vitro synthetic approaches, including modifications of solid phase peptide synthesis (SPPS) methods, relative to biologic and biomimetic approaches. First, the composition of the molecules is not limited to the normal set of physiological amino acids; it is possible to design amino acid analogs and incorporate them using well-established solid phase synthesis methods. Parallel synthesis can also be brought to bear, thereby dramatically increasing the number of substrate candidates. Because the approach is performed entirely in vitro, many of the concerns that arise from in vivo syntheses of bioactive molecules are eliminated. For example, degradation of products during fermentation would not be a concern, nor would the cytotoxic effects of the bioactive molecule on the producer microorganism be of concern.
In order to achieve the goal of in vitro synthesis, orthogonal lanthionines with potentially suitable protecting groups have been designed for SPPS using different approaches, such as the Michael addition of cysteine to preformed Dha. Probert et al., (1996) Tetrahedron Letters. 37, 1101-1104. This method led to a 1:1 mixture of diastereomers and, hence, was shown to have little commercial value. The ring opening of serine lactone with protected cysteines has also been reported but this led to a mixture of lanthionines and thioesters. The ring opening of aziridines has been investigated but was shown to produce regioisomeric mixtures due to opening of the aziridine at the α and β position. Dugave & Menez (1997) Tetrahedron-Asymmetry. 8, 1453-1465; Swali et al., (2002) Tetrahedron. 58, 9101-9109. More recent reports suggest that alkylating a suitably protected cysteine with a protected β-bromoalanine can result in the synthesis of lanthionines, but this method does not permit the construction of molecules with overlapping rings. Zhu (2003) European Journal of Organic Chemistry. 20, 4062-4072.
Because the Fmoc/Boc protected analogs that are commercially available for SPPS are not sufficient to solve the challenge of synthesizing lantibiotics and other conformationally contrained bioactive peptides, there exists a need in the art for the synthesis of peptides with intramolecular bridges that create internal ring structures, including multiple rings and overlapping ring structures. In particular, there exists a need for in vitro methods for synthesizing lantibiotics on a large scale.